The present invention pertains to the production of a crystalline salt of sn-glycerol-3-phosphate and to its subsequent use in the direct preparation of diacyl phosphatidic acids. More specifically, this invention relates to the production of the mono-N,N-dimethyl-4-aminopyridinium salt of sn-glycerol-3-phosphate (G-3-P(DMAP).sub.1), a uniquely crystalline and anhydrous form of the phosphate. In addition, this invention relates to the method of direct acylation of the aforementioned N,N-dimethyl-4-aminopyridinium sn-glycerol-3-phosphate salt with fatty acid anhydrides so as to provide new methods for production of diacyl phosphatidic acids.
Diacyl phosphatidic acids are useful precursors for the synthesis of phospholipids which are major components of cellular membranes. Diacyl phosphatidic acids may be prepared by the acylation of various salt forms of sn-glycerol-3-phosphate. For example, Y. Lapidot and Z. Selinger in J. Am. Chem. Soc., Vol 87, 5522-5523 (1965) described the synthesis of diacyl phosphatidic acids via the acylation of pyridinium sn-glycerol-3-phosphate. The Lapidot et al. method gave acceptable yields (70%-80%) of the desired diacyl phosphatidic acids without significant formation of by-products, but on a small scale (0.4 mmol). Gupta et al. in Proc. Nat. Acad. Sci., Vol. 74, 4315-4319 (1977) described an improvement on the method of Lapidot et al., wherein the basic catalyst, N,N-dimethyl-4-aminopyridine, was added to enhance the reactivity of the fatty acid anhydride towards nucleophilic attack by pyridinium sn-glycerol-3-phosphate. According to Gupta et al., pyridinium sn-glycerol-3-phosphate gave superior yields (87%) of diacyl phosphatidic acid when reacted with fatty acid anhydride (3 equiv) and N,N-dimethyl-4-aminopyridine (4 equiv). While the method of Gupta et al. is amenable for production of diacyl phosphatidic acids on a millimolar scale, there are serious limitations in the large-scale synthesis of diacyl phosphatidic acids using this method.
It is one aspect of the present invention to provide new methods for the synthesis of diacyl phosphatidic acids which are useful for large-scale application.
Another limitation to these conventional methods for diacyl phosphatidic acid production is the required use of pyridinium sn-glycerol-3-phosphate. As described by Gupta et al., pyridinium sn-glycerol-3-phosphate salt is a hygroscopic and gummy oil which consequently poses several disadvantages in large-scale acylation reactions. As an intractable and hygroscopic oil, the pyridinium sn-glycerol-3-phosphate is difficult to accurately weigh thereby making quantification of reaction stoichiometry problematic. The difficulty in completely removing water and alcohols from the pyridinium sn-glycerol-3-phosphate poses another significant disadvantage.
Because the reaction conditions for diacyl phosphatidic acid production must be free of water and alcohol, large-scale diacyl phosphatidic acid synthesis using pyridinium sn-glycerol-3-phosphate poses substantial disadvantages in order to render the salt anhydrous and solvent-free. Both the Lapidot et al. and the Gupta et al. methods are labor intensive and require repeated addition of dry pyridine and subsequent evaporation of the solvent to render the pyridinium sn-glycerol-3-phosphate salt anhydrous.
It is another aspect of the present invention to avoid the use of pyridinium sn-glycerol-3-phosphate in the synthesis of diacyl phosphatidic acid.
Another serious drawback to the methods of Lapidot et al. and Gupta et al. is the indirect formulation of the pyridinium sn-glycerol-3-phosphate itself. Conventional methods known to date provide for the derivation of pyridinium sn-glycerol-3-phosphate from other sn-glycerol-3-phosphate salt forms. The other known crystalline salts of sn-glycerol-3-phosphate are the barium, calcium, sodium, monocyclohexylammonium and the dicyclohexylammonium sn-glycerol-3-phosphates. Both the barium and dicyclohexylammonium salts are generally prepared solely as a means to isolate the sn-glycerol-3-phosphate (C. F. Crans et al. in J. Am. Chem. Soc., Vol 107, 7019 (1986)). However, neither salt may be used directly for diacyl phosphatidic acid production since the barium salt is insoluble in the requisite organic reaction medium and the dicyclohexylammonium salt is not compatible with the coreactant fatty-acid anhydrides. To make either the barium or cyclohexylammonium salts compatible it is necessary to convert them to the pyridinium salt of sn-glycerol-3-phosphate if an acylation reaction is to be conducted. Such a conversion is a laborious process requiring exchange of the counter-ion for pyridine via the use of an ion-exchange resin. The solvent used to convert the salt to the pyridinium sn-glycerol-3-phosphate must be aqueous pyridine. The subsequent isolation of the pyridinium sn-glycerol-3-phosphate from the aqueous solution and the drying of the salt is, however, too expensive in terms of time and effort to make this procedure practical on a kilogram scale.
It is yet another aspect of the present invention to avoid the use of barium and dicyclohexylammonium salts in the preparation of diacyl phosphatidic acids.
It is still yet another aspect of the present invention to provide a method to readily isolate a salt of sn-glycerol-3-phosphate in a crystalline and anhydrous form which can be subsequently directly acylated to economically form phosphatidic acids, and to provide a process for the synthesis of diacyl phosphatidic acids and other phospholipids from sn-glycerol-3-phosphate.